The ability to develop and sustain populations of memory CD8 T cells after infection or immunization is a hallmark of the adaptive immune response and one basis for protective vaccination against infectious disease or in cancer immunotherapy. Since the level of protection to infection is proportional to the number of memory CD8 T cells present at the time of pathogen exposure, many vaccines in use today involve an initial immunization that is followed by one or more booster ('prime-boost'protocols) immunizations. Similarly, individuals that receive only a single immunization may receive a secondary stimulation when they encounter the pathogen and, even in the absence of vaccination individuals might be exposed to multiple rounds of infection with the same pathogen. Therefore, there is a need to understand CD8 T cell responses generated after numerous antigen exposures. Importantly, most studies of memory CD8 T cells have focused on the response to primary (1o) infection or vaccination. This is a critical knowledge gap since it is not known how the number of Ag-exposures can influence the characteristics of CD8 T cell populations and their ability to defend against infection. Our long term goal is to fully understand the functional consequences imposed on CD8 T cell populations by multiple antigen encounters. This information will be significant in understanding how best to generate and manipulate protective immunity achieved by vaccination. We will test the central hypothesis that the number of antigen-stimulations will dictate the important phenotypic and functional characteristics of memory CD8 T cell populations. We are well prepared to undertake these studies based upon our expertise in mouse models of infections and CD8 T cell immunity coupled with the guidance of strong preliminary data. We have developed novel methodologies that will enable comprehensive and detailed analyses of CD8 T cell populations responding to multiple antigen encounters. Thus, we propose the following Specific Aims: SA1 - Determine how the number of antigen encounters controls the longevity of CD8 T cells. SA2 - Evaluate the phenotypic and functional differences between memory CD8 T cell populations in response to multiple antigen-stimulations. SA3 - Define the role of inflammation in generation of secondary (2o) and tertiary (3o) memory CD8 T cell responses. Public Health Relevance: Most studies of memory CD8 T cells have focused on the response to primary (1o) infection and/or vaccination. However, booster immunizations are often used to increase protective CD8 T cell numbers and are common feature of vaccines used to protect humans against infectious disease. The goal of this proposal, to fully characterize the functional consequences imposed on CD8 T cell populations by multiple rounds of Ag-stimulations, will be significant in understanding how best to generate protective immunity by vaccination.